Large-scale Isolation, Expansion and Characterization of Human Amniotic Epithelial Cells

Abstract

Background and ObjectivesThe human Amniotic epithelial cells (AME) derived from amniotic membrane of placenta have been considered as the potential fetal stem cell source with minimal or no ethical concerns and are important therapeutic tool for anti-fibrotic and regenerative therapies.Methods and ResultsHere, we evaluated the isolation, media screening, scale-up and characterization of AME cells. The isolation, expansion of AMEs were performed by sequential passaging and growth kinetics studies. The AMEs were characterized using immunocytochemistry, immunophenotyping, In-vitro differentiation, and anti-fibrotic assays. The growth kinetics study revealed that the AME cultured in Ultraculture (UC) and DMEM knockout (DMEM-KO) have prominently higher growth rate compared to others. Overall, the AMEs cultured from 5 different media retained basic morphological characteristics and the functional characteristics.ConclusionsOur result suggests that the AMEs can be successfully cultured in UC based complete media without losing its epithelial cell characteristics even after passaging for passage 2 (P2). However, a careful and methodical pre-clinical and clinical translation studies need to be conducted to show its safety and efficacy.

Producing Human Amniotic Epithelial Cells-only Membrane for Transplantation

“…In conventional methods, hAECs were isolated through multiple digestion by trypsin [15-21,37]. The isolated cells by conventional methods could contain more hAMSCs and have lower purity of hAECs.…”

“…In recent years, many studies had established methods of isolating hAECs [15-21]. However, the characteristic of these hAECs from different methods were quite different.…”

“…Then, these hAECs could happen epithelial-to-mesenchymal transition, or spontaneous differentiation, which were adverse to their clinical application. To address these problems, some studies used different factors such as progesterone, EGF or Supplement S7in hAECs’ culture [5,15-19,21,39,48]. However, these results were still not ideal.…”

“…One of the obstacles is that the purity of isolated hAECs still need improvement. In conventional methods, hAECs were isolated from hAM through multiple treatment of trypsin [15-21]. hAM are consist of three layers: single-layer of hAECs, a thick layer of BM which mainly made up of collagen and hyaluronic acid, and a thick layer of hAMSCs.…”

“…To address the problems mentioned above, we produced hAECs membrane of epithelium construction by collagenase and cell scraper instead of trypsin. In previous studies, collagenase had been repeatedly tested, compared with trypsin, and turned out to be inefficient in isolation of hAECs [18-21]. One of the main reasons could be that collagenase affected both the BM and hAMSCs.…”